Herpes virus strains for gene therapy

ABSTRACT

The present invention provides a herpes virus with improved oncolytic properties which comprises a gene encoding an immunomodulatory cytokine and which lacks a functional ICP34.5 gene and a functional ICP47 encoding gene.

This application is the US national phase of international applicationPCT/GB01/00225 filed 22 Jan. 2001, which designated the US and waspublished in English.

FIELD OF THE INVENTION

The present invention relates to herpes virus strains with improvedanti-tumour activity as compared to previously known strains.

BACKGROUND TO THE INVENTION

Viruses have been shown to have utility in a variety of applications inbiotechnology and medicine on many occasions. Each is due to the uniqueability of viruses to enter cells at high efficiency. This is followedin such applications by either virus gene expression and replicationand/or expression of an inserted heterologous gene. Thus viruses caneither deliver and express genes in cells (either viral or other genes)which may be useful in for example gene therapy or the development ofvaccines, or they may be useful in selectively killing cells by lyticreplication or the action of a delivered gene in for example cancer.

Herpes simplex virus (HSV) has been suggested to be of use for theoncolytic treatment of cancer. Here the virus must however be disabledsuch that it is no longer pathogenic, i.e. does not replicate in andkill non-tumor cells, but such that it can still enter and kill tumorcells. For the oncolytic treatment of cancer, which may also include thedelivery of gene(s) enhancing the therapeutic effect, a number ofmutations to HSV have been identified which still allow the virus toreplicate in culture or in actively dividing cells in vivo (e.g. intumors), but which prevent significant replication in normal tissue.Such mutations include disruption of the genes encoding ICP34.5, ICP6,and thymidine kinase. Of these, viruses with mutations to ICP34.5, orICP34.5 together with mutation of e.g. ICP6 have so far shown the mostfavourable safety profile. Viruses deleted for only ICP34.5 have beenshown to replicate in many tumor cell types in vitro and to selectivelyreplicate in artificially induced brain tumors in mice while sparingsurrounding tissue. Early stage clinical trials have also shown theirsafety in man.

However, while promise has been shown for various viruses including HSVfor the oncolytic treatment of cancer, the majority of this work hasused virus strains which do not carry a heterologous gene which mayenhance the anti-tumor effect. We propose that the combined use of HSVwith an inactivating mutation in the gene encoding ICP34.5 together withthe delivery of the gene encoding an immunomodulatory protein such asgranulocyte macrophage colony stimulating factor (GM-CSF) encoded in thedisabled virus genome may have optimal immune stimulating propertiesagainst the tumor to be treated, particularly if functions in the viruswhich usually reduce immune responses to HSV infected cells have alsobeen inactivated. For example the HSV ICP47 protein specificallyinhibits antigen presentation in HSV infected cells (Hill et al 1995),and the product of the UL43 gene and the vhs protein reduce theimmune-stimulating abilities of dendritic cells infected with HSV. ICP47and/or dendritic cell-inactivating genes might therefore usefully bedeleted from an oncolytic HSV mutant virus used for the treatment ofcancer, particularly if immune effects are to be enhanced through theuse of GM-CSF or other immunostimulatory cytokine or chemokine. GM-CSFhas recently been shown to give an enhanced anti-tumor immune effect ifexpressed from within a tumor cell rather than administered systemically(Shi et al 1999). Thus in such use an oncolytic HSV mutant would beinoculated into a primary or secondary tumor where replication andoncolytic destruction of the tumor would occur. Immune responses wouldalso be stimulated against the HSV infected cells, and also to tumorcells elsewhere which had spread from the primary tumor site.

SUMMARY OF THE INVENTION

The present invention provides viruses with improved capabilities forthe lytic destruction of tumor cells in vivo. Here herpes simplex virusstrains are constructed using a strain of HSV1 or HSV2 in which thegenes encoding ICP34.5 and ICP47 have been inactivated such that afunctional ICP34.5 or ICP47 protein cannot be expressed and which alsocarries a gene encoding an immunomodulatory protein. The virus may alsobe mutated for any additional gene(s) which may be involved ininhibiting the function of dendritic cells including the UL43 geneand/or the gene encoding vhs. The present invention therefore providesviruses capable of the oncolytic destruction of tumor cells and in whichanti-tumor immune effects will have been maximised.

Accordingly the invention provides:

-   -   a herpes virus which comprises a gene encoding an        immunomodulatory protein and which lacks functional ICP34.5 and        ICP47 encoding genes.    -   a herpes virus of the invention for use in a method of treatment        of the human or animal body by therapy.    -   use of a virus of the invention in the manufacture of a        medicament for the treatment of cancer.    -   a pharmaceutical composition comprising as active ingredient a        virus according to the invention and a pharmaceutically        acceptable carrier or diluent.    -   a method of treating a tumour in an individual in need thereof        by administering to said individual an effective amount of a        virus according to the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Viruses

From top to bottom, diagrams show: laboratory HSV1 strain 17+, clinicalHSV1 strain JS1, strain 17+/ICP34.5−, strain JS1/ICP34.5−, strainJS1/ICP34.5−/ICP47−/hGMCSF, strain JS1/ICP34.5−/ICP47−/mGMCSF.

DETAILED DESCRIPTION OF THE INVENTION

A. Viruses

A herpes virus of the invention is capable of efficiently infectingtarget tumor cells and the genes encoding ICP34.5 and ICP47 areinactivated in the virus. Mutation of ICP34.5 allows selective oncolyticactivity. Such mutations are described in Chou et al 1990 and Maclean etal 1991, although any mutation in which ICP34.5 is non-functional may beused. The genes encoding ICP6 and/or thyridine kinase may additionallybe inactivated, as may other genes if such inactivation doessignificantly reduce the oncolytic effect, or if such deletion enhancesoncolytic or other desirable properties of the virus. ICP47 usuallyfunctions to block antigen presentation in HSV-infected cells so itsdisruption leads to a virus that does not confer on infected tumourcells properties that might protect them from the host's immune systemwhen infected with HSV. Viruses of the invention additionally encode animmunomodulatory protein, preferably GM-CSF, but may also encode othercytokines, chemokines such as RANTES, or other immunemodulatory proteinssuch as B7. 1. B7.2 or CD40L. Genes encoding immunomodulatory proteinsmay be included individually or in combination.

Viral regions altered for the purposes described above may be eithereliminated (completely or partly), or made non-functional, orsubstituted by other sequences, in particular by a gene for animmunomodulatory protein such as GM-CSF.

The virus of the invention may be derived from a HSV1 or HSV2 strain, orfrom a derivative thereof, preferably HSV1. Derivatives includeinter-type recombinants containing DNA from HSV1 and HSV2 strains. Suchinter-type recombinants are described in the art, for example inThompson et al, 1998 and Meignier et al, 1988. Derivatives preferablyhave at least 70% sequence homology to either the HSV1 or HSV2 genomes,more preferably at least 80%, even more preferably at least 90 or 95%.More preferably, a derivative has at least 70% sequence identity toeither the HSV1 or HSV2 genome, more preferably at least 80% identity,even more preferably at least 90%, 95% or 98% identity.

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings)(Devereux et al. (1984) Nucleic Acids Research 12, p387–395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290–300; Altschul et al.(1990) J. Mol. Biol. 215:403–10.

Software for performing BLAST analyses is publicly available through theNational Centre for Biotechnology Information's website. This algorithminvolves first identifying high scoring sequence pair (HSPs) byidentifying short words of length W in the query sequence that eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighbourhood word score threshold (Altschul et al., 1990). Theseinitial neighbourhood word hits act as seeds for initiating searches tofind HSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Nail. Acad. Sci. USA 89: 10915–10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci.USA 90: 5873–5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

A derivative may have the sequence of a HSV1 or HSV2 genome modified bynucleotide substitutions, for example from 1, 2 or 3 to 10, 25, 50 or100 substitutions. The HSV1 or HSV2 genome may alternatively oradditionally be modified by one or more insertions and/or deletionsand/or by an extension at either or both ends.

Virus strains of the invention may be “non-laboratory” strains. Thesecan also be referred to as “clinical” strains. A person of skill in theart will readily be able to distinguish between a laboratory strain anda non-laboratory, or clinical, strain. Further guidance on theproperties likely to be exhibited by virus strains is given below.

The key distinction between a laboratory and non-laboratory strain isthat laboratory strains currently in common use have been maintained forlong periods, many years in some cases, in culture. The culture ofviruses such as HSV involves a technique known as serial passage. Togrow and maintain viruses, suitable cells are infected with the virus,the virus replicates within the cell and the virus is then harvested;fresh cells are then re-infected, this process constitutes one cycle ofserial passage. Each such cycle may take, for example, a few days in thecase of HSV. As discussed above, such serial passaging may lead tochanges in the properties of the virus strain, in that selection takesplaces for properties that favour growth in culture (e.g. rapidreplication), as opposed to properties useful for practicalapplications, e.g. maintenance of the capacity to travel along axons inthe case of HSV or to infect human cells.

Virus strains of the invention are may be non-laboratory strains in thatthey are derived from strains recently isolated from infectedindividuals. Strains of the invention are modified compared to theoriginal clinical isolates, and may have spent a time in culture, butany time spent in culture will be comparatively short. Strains of theinvention are prepared in such a manner as to retain substantially thedesirable properties of the original clinical isolates from which theyare derived.

A virus strain of the invention is derived from a parental virus strainif the parental virus strain is mutated to produce the virus. Forexample, a virus of the invention may be derived from the clinicalisolate JSI. The parental strain of such a JSI-derived virus may be JSIor another HSV1 strain derived from JSI. Thus a virus of the inventionmay be a JSI virus comprising a gene encoding an immunomodulatoryprotein and which lacks a functional ICP34.5 encoding gene and afunctional ICP47 encoding gene. In addition, such a virus may containany other mutation as mentioned herein.

A virus of the invention is capable of efficiently infecting targethuman cancer cells. When such a virus is a non-laboratory or clinicalstrain it will have been recently isolated from an HSV infectedindividual and then screened for the desired ability of enhancedreplication, infection or killing of tumour and/or other cells in vitroand/or in vivo in comparison to standard laboratory strains. Suchviruses of the invention with improved properties as compared tolaboratory virus strains are then engineered such that they lackfunctional ICP34.5 and ICP47 genes and encode a gene(s) for animmunomodulatory protein(s) such as GM-CSF under the control of asuitable promoter(s). Other genes encoding proteins which interfere withthe function of dendritic cells such as UL43 and/or vhs may also beinactivated.

Preferably, a non-laboratory virus strain of the invention has undergonethree years or less in culture since isolation of its unmodifiedclinical precursor strain from its host. More preferably, the strain hasundergone one year or less in culture, for example nine months or less,six months or less, three months or less, or two months or less, onemonth or less, two weeks or less., or one week or less. By thesedefinitions of time in culture, is meant time actually spent in culture.Thus, for example, it is a common practice to freeze virus strains inorder to preserve them. Evidently, preserving by freezing or in anequivalent manner does not qualify as maintaining the strain in culture.Thus, time spent frozen or otherwise preserved is not included in theabove definitions of time spent in culture. Time spent in culture istypically time actually spent undergoing serial passage, i.e. timeduring which selection for undesirable characteristics can occur.

Preferably, a non-laboratory virus strain has undergone 1,000 or lesscycles or serial passage since isolation of its unmodified clinicalprecursor strain from its host. More preferably, it has undergone 500 orless, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 orless, 40 or less, 30 or less, 20 or less, or 10 or less such cycles.

Preferably, a non-laboratory virus has a greater ability, as measured bystandard statistical tests, than a reference laboratory strain with theequivalent modifications to perform certain functions useful in theapplication at hand. In the case of an oncolytic virus for tumourtreatment, a non-laboratory virus strain of the invention willpreferably have a greater ability than a reference laboratory strainwith equivalent modifications to infect or replicate in tumour cells, tokill tumour cells or to spread between cells in tissue. More preferably,such greater ability is a statistically significantly greater ability.For example, according to the invention, a non-laboratory strain of theinvention may have up to 1.1 fold, 1.2 fold, 1.5 fold, 2 fold, 5 fold,10 fold, 20 fold, 50 fold, or 100 fold the capacity of the referencestrain in respect of the property being tested.

Statistical analysis of the properties described herein may be carriedout by standard tests, for example, t-tests, ANOVA, or Chi squaredtests. Typically, statistical significance will be measured to a levelof p=0.05 (5%), more preferably p=0.01 p, p=0.001, p=0.0001, p=0.000001.

Viruses of the invention infect and replicate in tumour cells,subsequently killing the tumour cells. Thus, such viruses arereplication competent. Preferably, they are selectively replicationcompetent in tumour cells. This means that either they replicate intumour cells and not in non-tumour cells, or that they replicate moreeffectively in tumour cells than in non-tumour cells. Cells in which thevirus is able to replicate are permissive cells. Measurement ofselective replication competence can be carried out by the testsdescribed herein for measurement of replication and tumour cell-killingcapacity, and also analysed by the statistical techniques mentionedherein if desired.

A virus of the invention preferably has a greater ability than anunmodified parent strain to infect or replicate in a tumour cell, tokill tumour cells or to spread between cells in tissues. Preferably thisability is a statistically significant greater ability. For example, avirus according to the invention may have up to 1.1 fold, 1.2 fold, 1.5fold, 2 fold, 5 fold, 10 fold, 20 fold, 50 fold or 100 fold the capacityof the unmodified parent strain in respect of the property being tested.

The properties of the virus strain in respect of tumour cells can bemeasured in any manner known in the art. For example, the capacity of avirus to infect a tumour cell can be quantified by measuring the dose ofvirus required to measure a given percentage of cells, for example 50%or 80% of cells. The capacity to replicate in a tumour cell can bemeasured by growth measurements such as those carried out in theExamples, e.g. by measuring virus growth in cells over a period of 6,12, 24, 36, 48 or 72 hours or longer.

The ability of a virus to kill tumour cells can be roughly quantitatedby eye or more exactly quantitated by counting the number of live cellsthat remain over time for a given time point and MOI for given celltype. For example, comparisons may be made over 24, 48 or 72 hours andusing any known tumour cell type. In particular, HT29 colorectaladenocarcinoma, LNCaP.FGC prostate adenocarcinoma, MDA-MB-231 breastadenocarcinoma, SK-MEL-28 malignant melanoma or U-87 MG glioblastomaastrocytoma cells can be used. Any one of these cell types or anycombination of these cell types can be used, as may other tumour celltypes. It may be desirable to construct a standard panel of tumour celltypes for this purpose. To count the number of live cells remaining at agiven time point, the number of trypan blue-excluding cells (i.e. livecells) can be counted. Quantitation may also be carried out byfluorescence activated cell sorting (FACS) or MTT assay. Tumourcell-killing ability may also be measured in vivo, e.g. by measuring thereduction in tumour volume engendered by a particular virus.

In order to determine the properties of viruses of the invention, itwill generally be desirable to use a standard laboratory referencestrain for comparison. Any suitable standard laboratory reference strainmay be used. In the case of HSV, it is preferred to use one or more ofHSV1 strain 17+, HSV1 strain F or HSV1 strain KOS. The reference strainwill typically have equivalent modifications to the strain of theinvention being tested. Thus, the reference strain will typically haveequivalent modifications gene deletions and, such as heterologous geneinsertions. In the case of a virus of the invention, where the ICP34.5and ICP47-encoding genes have been rendered non-functional, then theywill also have been rendered nonfunctional in the reference strain. Themodifications made to the reference strain may be identical to thosemade to the strain of the invention. By this, it is meant that the genedisruptions in the reference strain will be in exactly equivalentpositions to those in the strain of the invention, e.g. deletions willbe of the same size and in the same place. Similarly, in theseembodiments, heterologous genes will be inserted in the same place,driven by the same promoter, etc. However, it is not essential thatidentical modifications be made. What is important is that the referencegene has functionally equivalent modifications, e.g. that the same genesare rendered nonfunctional and/or the same heterologous gene or genes isinserted.

B. Methods of Mutation

The various genes referred to may be rendered functionally inactive byseveral techniques well known in the art. For example, they may berendered functionally inactive by deletion(s), substitution(s) orinsertion(s), preferably by deletion. Deletions may remove one or moreportions of the gene or the entire gene. For example, deletion of onlyone nucleotide may be made, resulting in a frame shift. However,preferably a larger deletion( ) is made, for example at least 25%, morepreferably at least 50% of the total coding and non-coding sequence (oralternatively, in absolute terms, at least 10 nucleotides, morepreferably at least 100 nucleotides, most preferably, at least 1000nucleotides). It is particularly preferred to remove the entire gene andsome of the flanking sequences. Where two or more copies of the gene arepresent in the viral genome it is preferred that both copies of the geneare rendered functionally inactive.

Mutations are made in the herpes viruses by homologous recombinationmethods well known to those skilled in the art. For example, HSV genomicDNA is transfected together with a vector, preferably a plasmid vector,comprising the mutated sequence flanked by homologous HSV sequences. Themutated sequence may comprise a deletion(s), insertion(s) orsubstitution(s), all of which may be constructed by routine techniques.Insertions may include selectable marker genes, for example lacZ orgreen fluorescent protein (GFP), for screening recombinant viruses, forexample, β-galactosidase activity or fluorescence.

C. Heterologous Genes and Promoters

The viruses of the invention may be modified to carry a heterologousgene encoding an immunomodulatory protein. Preferably theimmunomodulatory protein will enhance the anti-tumour activity of thevirus. More preferably the protein is GM-CSF or another cytokine, achemokine such as RANTES, or another immunomodulatory molecule such asB7.1, B7.2 or CD40L. Most preferably the immunomodulatory molecule isGM-CSF. The immunomodulatory gene may be any allelic variant of awild-type gene, or it may be a mutant gene. The immunomodulatory genewill be derived from a mammal, preferably a rodent or primate, morepreferably a human. The immunomodulatory gene is preferably operablylinked to a control sequence permitting expression of said gene in acell in vivo. Viruses of the invention may thus be used to deliver theimmunomodulatory gene (or genes) to a cell in vivo where it will beexpressed.

The immunomodulatory gene may be inserted into the viral genome by anysuitable technique such as homologous recombination of HSV strains with,for example, plasmid vectors carrying the gene flanked by HSV sequences.The GM-CSF gene, or other immunomodulatory gene, may be introduced intoa suitable plasmid vector comprising herpes viral sequences usingcloning techniques well-known in the art. The gene may be inserted intothe viral genome at any location provided that oncolytic properties arestill retained. Immunomodulatory genes may be inserted at multiple siteswithin the virus genome. For example, from 2 to 5 genes may be insertedinto the genome.

The transcribed sequence of the immunomodulatory gene is preferablyoperably linked to a control sequence permitting expression of the genein a tumour cell. The term “operably linked” refers to a juxtapositionwherein the components described are in a relationship permitting themto function in their intended manner. A control sequence “operablylinked” to a coding sequence is ligated in such a way that expression ofthe coding sequence is achieved under conditions compatible with thecontrol sequence.

The control sequence comprises a promoter allowing expression of theimmunomodulatory gene and a signal for termination of transcription. Thepromoter is selected from promoters which are functional in mammalian,preferably human tumour cells. The promoter may be derived from promotersequences of eukaryotic genes. For example, the promoter may be derivedfrom the genome of a cell in which expression of the heterologous geneis to occur, preferably a mammalian, preferably a human tumour cell.With respect to eukaryotic promoters, they may be promoters thatfunction in a ubiquitous manner (such as promoters of β-actin, tubulin)or, alternatively, in a tumour-specific manner. They may also bepromoters that respond to specific stimuli, for example promoters thatbind steroid hormone receptors. Viral promoters may also be used, forexample the Moloney murine leukaemia virus long terminal repeat (MMLVLTR) promoter or other retroviral promoters, the human or mousecytomegalovirus (CMV) IE promoter, or promoters of herpes virus genesincluding those driving expression of the latency associatedtranscripts.

Expression cassettes and other suitable constructs comprising theimmunomodulatory gene and control sequences can be made using routinecloning techniques known to persons skilled in the art (see, forexample, Sambrook et al., 1989, Molecular Cloning—A laboratory manual;Cold Spring Harbor Press).

It may also be advantageous for the promoters to be inducible so thatthe levels of expression of the immunomodulatory gene(s) can beregulated during the life-time of the tumour cell. Inducible means thatthe levels of expression obtained using the promoter can be regulated.For example, a virus of the invention may further comprise ahererologous gene encoding the tet repressor/VP16 transcriptionalactivator fusion protein under the control of a strong promoter (e.g.the CMV IE promoter) and the immunomodulatory gene may be under thecontrol of a promoter responsive to the tet repressor VP 16transcriptional activator fusion protein previously reported (Gossen andBujard, 1992, Gossen et al, 1995). Thus, in this example, expression ofthe immunomodulatory gene would depend on the presence or absence oftetracycline.

Multiple heterologous genes can be accommodated in the herpes virusgenome. Therefore, a virus of the invention may comprise two or moreimmunomodulatory genes, for example from 2 to 3, 4 or 5 immunomodulatorygenes. More than one gene and associated control sequences could beintroduced into a particular HSV strain either at a single site or atmultiple sites in the virus genome. Alternatively pairs of promoters(the same or different promoters) facing in opposite orientations awayfrom each other, each driving the expression of an immunomodulatory genemay be used.

D. Therapeutic Uses

Viruses of the invention may be used in methods of cancer therapy of thehuman or animal body. In particular, viruses of the invention may beused in the oncolytic treatment of cancer, either with or withoutadditional pro-drug therapy or stimulation of an anti-tumour immuneresponse. Viruses of the invention may be used in the therapeutictreatment of any solid tumour in a mammal, preferably in a human. Forexample viruses of the invention may be administered to a subject withprostate, breast, lung, liver, endometrial, bladder, colon or cervicalcarcinoma; adenocarcinoma; melanoma; lymphoma; glioma; or sarcomas suchas soft tissue and bone sarcomas.

E. Administration

The viruses of the invention may be used in a patient, preferably ahuman patient, in need of treatment. A patient in need of treatment isan individual suffering from cancer, preferably an individual with asolid tumour. The aim of therapeutic treatment is to improve thecondition of a patient. Typically therapeutic treatment using a virus ofthe invention allieviates the symptoms of the cancer. A method oftreatment of cancer according to the invention comprises administering atherapeutically effective amount of a virus of the invention to apatient suffering from cancer. Administration of an oncolytic virus ofthe invention to an individual suffering from a tumour will typicallykill the cells of the tumour thus decreasing the size of the tumourand/or preventing spread of malignant cells from the tumour.

One method of administering therapy involves combining the virus with apharmaceutically acceptable carrier or diluent to produce apharmaceutical composition. Suitable carriers and diluents includeisotonic saline solutions, for example phosphate-buffered saline.

Therapeutic treatment may be carried out following direct injection ofthe virus composition into target tissue which may be the tumour or ablood vessel supplying the tumour. The amount of virus administered isin the case of HSV in the range of from 10⁴ to 10¹⁰ pfu, preferably from10⁵ to 10⁸ pfu, more preferably about 10⁶ to 10⁸ pfu. Typically up to500 μl, typically from 1 to 200 μl preferably from 1 to 10 μl of apharmaceutical composition consisting essentially of the virus and apharmaceutically acceptable suitable carrier or diluent would be usedfor injection. However for some oncolytic therapy applications largervolumes up to 10 ml may also be used, depending on the tumour and theinoculation site.

The routes of administration and dosages described are intended only asa guide since a skilled practitioner will be able to determine readilythe optimum route of administration and dosage. The dosage may bedetermined according to various parameters, especially according to thelocation of the tumour, the size of the tumour, the age, weight andcondition of the patient to be treated and the route of administration.Preferably the virus is administered by direct injection into thetumour. The virus may also be administered systemically or by injectioninto a blood vessel supplying the tumour. The optimum route ofadministration will depend on the location and size of the tumour.

The following Examples illustrates the invention.

Herpes simplex type-1 virus (HSV1) in which the neurovirulence factorICP34.5 is inactivated has previously been shown to direct tumourspecific cell lysis in tumour models both in vitro and in vivo. Suchviruses have also been shown to be safe in Phase I clinical trials bydirect intra-cerebral injection in late stage glioma patients.

Previous work has used serially passaged laboratory isolates of HSV1(viruses derived from HSV1 strain 17+ or HSV1 strain F) which might beanticipated to be attenuated in their lytic capability in human tumourcells as compared to more recent clinical isolates.

In work aimed at producing ICP34.5 deleted HSV with enhanced oncolyticand anti-tumour potential, we have deleted ICP47 and ICP34.5 from HSV1strain JS1 and have inserted the immunomodulatory gene for GM-CSF.

Virus Construction (see FIG. 1)

The viruses used were either based on HSV1 strain 17+ (a standardlaboratory strain) or a clinical isolate derived from cold sores from afrequent re-activator of HSV1. This clinical, or “non-laboratory”,strain is named JS1. ICP34.5 was completely deleted from strain 17+ andJS 1 together with the insertion of a CMV-GFP cassette. JS1 was thenalso further engineered by the insertion of human GM-CSF (hGM-CSF) ormouse GM-CSF (mGM-CSF) so as to replace the ICP34.5 gene and by thedeletion of ICP47. The derivatives of JS1 discussed herein are alsonon-laboratory strains, i.e. modified non-laboratory strains of theinvention.

Lytic Capabilities of Viruses

Lytic (cell killing) capabilities were enhanced with the JS1-derivednon-laboratory strains derived virus in all tumour cell lines tested ascompared with the 17+ derived strains. More particularly, the JS1/34.5−virus, i.e. JS1 with ICP34.5 removed by deletion, showed enhanced lyticcapabilities in HT29 colorectal adenocarcinoma, LNCaP.FGC prostateadenocarcinoma, MDA-MB-231 breast adenocarcinoma, SK-MEL-28 malignantmelanoma and U-87 MG glioblastoma astrocytoma cells.

Thus, to provide increased oncolytic activity, the use of recentclinical virus strains is likely to enhance the anti-tumour capabilitiesof such viruses when used in human patients for cancer treatment.

Further Enhanced Anti-Tumour Activity

Further enhanced activity may also be anticipated if these viruses arethen used to deliver genes with anti-tumour activity. Such genes includethose encoding pro-drug activators or immunostimulatory proteins.

An ICP34.5 deleted clinical isolated of HSV1 which expresses human ormouse GM-CSF was produced from JS1. GM-CSF is a potent immunestimulator. These virus are designed to enhance anti-tumour immuneresponses following intra-tumoral injection. These viruses weredemonstrated to express human or mouse GM-CSF using ELISA assay kits(Biotrak, Amersham) when the viruses are produced in BHK cells inculture. Individual wells of a six well plate produced 0.56 or 0.54microgrammes of human or mouse GM-CSF respectively 24 hrs afterinfection of confluent BHK cells at MOI=0.5.

Deposit Information

HSV1 strain JS1 has been deposited at the European Collection of CellCultures (ECACC), CAMR, Sailsbury, Wiltshire SP4 PJG, United Kingdom, on2 Jan. 2001 under accession number 01010209.

REFERENCES

-   Hill et al. 1995, Nature 375; 411–415-   Shi et al. 1999, Cancer-Gene-Ther 6: 81–88-   Chou et al. 1990, Science 250: 1262–1266-   Maclean et al. 1991, J. Gen. Virol. 72: 631–639-   Gossen M & Bujard H, 1992, PNAS 89: 5547–5551-   Gossen M et al. 1995, Science 268: 1766–1769-   Thompson et al. 1998, Virus Genes 1(3); 275–286-   Meignier et al. 1988, Infect. Dis. 159; 602–614

1. A herpes simplex virus which: (i) comprises a gene encoding animmunostimulatory protein; (ii) lacks a functional ICP34.5 encoding geneand a functional ICP47 encoding gene; (iii) is replication competent intumor cells; and (iv) is derived from HSV1 JS1 as deposited at theEuropean collection of cell cultures (ECAAC) under accession number01010209.
 2. A virus according to claim 1, wherein saidimmunostimulatory protein is GM-CSF.
 3. A pharmaceutical compositioncomprising as active ingredient a virus according to claim 1 and apharmaceutically acceptable carrier or diluent.
 4. HSV1 strain JS1 asdeposited at the European Collection of Cell Cultures (ECACC) underaccession number 01010209, or an HSV1 strain derived therefrom.